Hydrophilic diagnostic devices for use in the assaying of biological fluids

ABSTRACT

Diagnostic in-vitro devices for use in the assaying of biological fluids are provided which include cover plates or backing strips which exhibit hydrophilic properties to assist in transport of the biological fluid or retention of same within the device. Exemplary diagnostic devices include lateral flow devices, microfluidic devices and microtiter plates. The devices may also be comprised of low fluorescent material in order to facilitate any diagnostic determination by use of fluorescent emissions. Hydrophilic properties may be imparted to the cover plates or backing strips by physical or chemical treatment thereof. The cover plates or backing strips may exhibit heat sealable or pressure sensitive properties.

BACKGROUND OF THE INVENTION

This application is directed to novel hydrophilic constructions forin-vitro diagnostic test devices.

Lateral flow test strips are routinely used in medical and otherapplications to provide convenient and simple analysis of many importantchemicals. S. M. Rosen, “Biomarkers of chemical exposure: A new Frontierin Clinical Chemistry”, IVD Technology, May (1996) p. 22; R. A.Esposito, A. T. Culliford, S. B. Colvin et al., “The Role of theActivated Clotting Time in Herparin Administration and Neutralizationfor Cardiopulmonary Bypass”, J. Thor. Card. Surg. 85 (1983), 174-185; C.A. McDonald, P. Syribeys, B. Hazelton, P. Bethea, T. Rigl, S. Hydro, S.J. Kennedy, 93^(rd) General Meeting of American Society Microbiology, “Arapid 1-step colored particle lateral flow immunoassay for the detectionof Group 1 Streptococcal Antigen extracted directly from Throat Swats”,93 (1993), p. 507; and C. Huang and E. Fan, “One StepImmunochromatographic Device and Method of Use”, U.S. Pat. No.5,712,172; A. Pronovost and J. Pawlak, “One Step Urine Creatine Assays”,U.S. Pat. No. 5,804,452.

Microtiter plates are used in the handling of liquid material samplesduring analytical assays for multiple, low volume analysis. Such platesinvolve the use of an assay plate having multiple depressions or wells,which provide a rapid automated analysis. Typically, such well have acapacity of 1 microliter. Such microliter plates have a variety of uses,including enzyme assays, receptor-ligand assays, cell based assays, etc.The use of such microliter plates may be either batch-wise, orcontinuous.

The use of a continuous strip of material having sample wells moldedalong the length of the strip of material is disclosed in U.S. Pat. No.4,883,642. This patent discloses means to automatically hold, process,store and analyze biological samples comprised of a ribbon provided withmicrowells for analysis of multiple samples. The microwells in theribbon may be protected by an adhered protective film or skin.

Microfluidic devices are also commonly-used in the assaying ofbiological samples. Such devices comprise a base platform within whichare formed a number of capillaries which serve to transport the samplefrom a receiving portion of the device to a collection portion.

All of the above diagnostic devices are well-known to those skilled inthe art.

In-vitro diagnostic devices are used to detect analytes such asnutrients, hormones, therapeutic drugs, drugs-of-abuse and environmentalcontaminates. In medical diagnostic test devices, biological fluids suchas whole blood, plasma, serum, nasal secretions, sputum, saliva, urine,sweat, transdermal exudates, cerebrospinal fluids and the like may beanalyzed for specific components that are clinically important formonitoring and diagnosis. In addition, microbiological suspensions andtissues may be homogenized in compatible liquids and the fluid analyzedfor specific components. Typically, the specimen fluid is deposited atan inlet port of a suitable in-vitro diagnostic test strip and thesample fluid is drawn into the device by mechanical means such as vacuumor by capillary flow action.

In-vitro diagnostic devices are used in various settings includinghospitals, clinics, alternative care sites and in the home. Thesedevices have been developed by various manufacturers to enable clinicalprofessionals and non-professionals to make accurate decisions for thediagnosis and management of medical conditions. Point-of-care devicessuch are used to analyze blood chemistry such as electrolytes and pH inboth clinical and non-clinical locations. Home pregnancy test kits areused to monitor hcG in urine. Diabetics routinely use diagnostic teststrips to monitor blood glucose concentrations. Amira Medical, “GlucoseMonitor without Fingersticking”, IVD Technology, July 1999, p. 16.

A number of U.S. and foreign patents describe the use of lateral flowassay devices. U.S. Pat. No. 5,798,273 and corresponding European patent833159 describe a direct read lateral flow device for detecting smallanalytes. WO 97/38126 describes a lateral flow device for measuringanalytes in whole blood. U.S. Pat. No. 5,804,452 describes a device forthe detection of creatinine in biological fluids such as urine in a onestep lateral flow sensor. U.S. Pat. No. 5,916,521 describes a verticalflow diagnostic device for the testing of body fluids. WO 99/34191describes a lateral flow test strip for the detection of an analyte suchas beta lactam in milk. See also, U.S. Pat. Nos. 4,857,453; 5,087,556;5,137,808; 5,712,170; 5,712,172; 5,804,452; 5,821,073; 5,985,675;5,989,921; 6,087,175 and 6,103,536.

Various types of capillary flow type diagnostic devices are also knownand have been used for some time. Exemplary of such devices are thoseshown in U.S. Pat. Nos. 6,048,498 and 6,117,395.

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the present invention to provide diagnostic deviceswhich enable benefits to be achieved not achieved by prior art devices.

It is an object of the present invention to provide lateral flow deviceswhich provide faster and more uniform flow of the sample, uniformwicking of membranes and a uniform capture line.

It is an object of the present invention to provide a microfluidicdevice which provides uniform wetting and wicking, ease of manufacture,which is non-contaminating to the sample, exhibits controlledevaporation and enables separation of components to be achieved.

It is further an object of the present invention to provide a microtiterplate diagnostic device which exhibits improved wetting, reducescondensation accumulation and serves to enhance the desired diagnostictesting.

In accordance with the above, there is provided a lateral flow in-vitrodiagnostic device comprising a housing, means in the housing tointroduce a sample to be assayed in the device, means in the housing forfluid collection, and a backing strip having spaced apart first andsecond ends, the improvement wherein the surface of the backing strip ishydrophilic in character.

In accordance with another embodiment of the invention, there isprovided a microfluidic in-vitro diagnostic device comprised of a basehaving at least one fluid channel within which a fluid sample to beassayed passes from an inlet port to a detection zone, with said atleast one fluid channel being sealed by an enclosure surface, theimprovement wherein at least one surface of the at least one fluidchannel is hydrophilic in character.

In accordance with still another embodiment of the invention, there isprovided a microfluidic in-vitro diagnostic device comprised of opposingbase portions separated by an adhesive spacer portion having fluidchannels therein within which a fluid to be assayed passes from an inletport to a detection zone, wherein at least a portion of the surfaces ofsaid base portions and said spacer portion defining said channel beinghydrophilic in character.

In accordance with the present invention, there is also provided amicroplate comprised of a base and having a multitude of microholes orcavities and at least one cover placed in sealing relationship to saidmicroholes or cavities and having a surface facing the interior of themicroholes or cavities which is hydrophilic in character.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a top view of a prior art lateral flow device.

FIG. 1 b is a schematic diagram of the prior art lateral flow device ofFIG. 1 a;

FIG. 2 is a depiction of capillary rise in a cylinder;

FIG. 3 is a depiction of the wetting of a fluid on a smooth flatsurface;

FIG. 4 graphically depicts the effect of surface treatment on contactangle;

FIG. 5 depicts a laboratory coating technique for casting adhesive on afilm;

FIG. 6 depicts a method for contact angle measurement on a flat surface;

FIG. 7 depicts a microfluidic device used in in-vitro sample analysis;

FIG. 8 depicts the effect of surfactant concentration on contact angle;

FIG. 9 depicts water contact angle vs. spreading time for hydrophilicfilms;

FIG. 10 depicts water contact angle vs. surfactant concentration forfilms;

FIG. 11 depicts the effect of surfactant concentration on contact angleand flow rate;

FIG. 12 is a side view of a lateral flow diagnostic device of thepresent invention;

FIG. 13 is a top view of the lateral flow diagnostic device of FIG. 12;

FIG. 14 is an exploded view of a lateral flow diagnostic test strip ofthe present invention;

FIG. 15 is an exploded view of another embodiment of a lateral flow teststrip of the present invention;

FIG. 16 is a view in perspective of a microfluidic diagnostic deviceaccording to the present invention;

FIG. 17 is a cross-sectional view of the device of FIG. 16;

FIG. 18 is a view in perspective of another embodiment of a microfluidicdevice having an adhesive spacer portion attached to a base portion;

FIG. 19 is a view in cross-section of the microfluidic device of FIG. 18wherein both base portions are present;

FIG. 20 is a view in perspective of a micro plate without a over sheet;and

FIG. 21 is a view in perspective of the micro plate of FIG. 18 with acover sheet.

FIG. 22 is a top view of an open well microplate having a multitude ofholes therein.

FIG. 23 is a view in cross-section of the open well microplate of FIG.22.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, adhesives and polymer filmsmay be formulated using polymer resins and surfactants to providemultifunctional bonding properties for use in in-vitro diagnosticdevices.

Hydrophilic adhesives or films may be formulated to be thermally bondedor pressure sensitive. The hydrophilicity of the surface of the adhesiveor film is controllable through the chemical structure, concentrationand distribution of the surfactant in the adhesive coating. Thehydrophilic properties reduce the surface tension of biological fluids(e.g., blood, urine, and sputum), thus allowing the rapid transfer offluid from an inlet area to a remote reagent area in an in-vitrodiagnostic device.

The invention will be described in connection with the Figures.

Lateral flow devices as shown in FIG. 1 typically have a sample inletarea for receiving the biological fluid. The sample inlet area or portmay be proximal to a conjugate pad that holds reagents specific to theanalytical test method. As the sample specimen flows from the inlet areathrough a reagent area, specific chemical reactions or a complexformation occur. The reaction product or complex continues to flow to adetection area where the analyte is monitored. Specimen fluids maycontinue to flow and be collected in an absorbent pad. The time requiredfor determining the concentration of a specific analyte is dependent onthe flow rate of the fluid and the reaction rate between the analyte anda specific test reagent.

Adhesive backings are typically used in the construction of lateral flowdevices to support the various components of the device including theconjugate pad, a microporous membrane with specific reagents and anabsorbent pad as shown in FIG. 1. The adhesive layer may be eitherpressure sensitive or heat-sealable, and may be present on a backingfilm such as a polyester film. The flow rate of the sample fluid istypically controlled by capillary flow through the microporous membrane.

Membranes used in lateral flow devices are typically hydrophobicpolymers with low surface energy. These membranes are polymers such asnitrocellulose, nylon, polyether sulfone, polyvinylidiene, and the like.Consequently, these components are not compatible with aqueousbiological fluids. To overcome the low surface energy of the membrane,surface active agents such as sodium dodecylsulfate (SDS) and sodiumdodecylbenzene sulfonate (SDBS) are added to increase the wettabilityand consequent wicking ability of the membrane. Although the addition ofsurface active agents to the membrane increases its wettability thesechemicals decrease the ability of the membrane to bond or retainproteins which may be critical to the analytical requirements and deviceperformance. In addition, surfactants added to the membrane can reducetest sensitivity by reducing signal intensity due to extensive spreadingof reagent bands.

It is known that the use of adhesives in diagnostic devices such aslateral flow devices to bond the hydrophilic membrane to the backinglayer can result in a reduction in the effectiveness of the hydrophilicmembrane layer during transport of the sample to be assayed across themembrane layer. Jones et al, IVD Technology, pp. 57-63, September, 2000.This reduced efficiency can be attributed to the migration of theadhesive into the membrane layer, creating isolated hydrophobic areaswithin the hydrophilic membrane. This effect is particularly enhancedupon use of an adhesive exhibiting low hardness (which exhibits highcold flow properties) in combination with a hydrophilic membrane ofminimal thickness, thus enhancing the ability of the adhesive to affectthe surface properties of the membrane. This effect can be minimized bythe use of high hardness adhesives which exhibit low cold flowproperties. However, high hardness adhesives also exhibit undesirablelower initial bond strength than a low hardness adhesive, a factor thatmust be taken into account when constructing the lateral flow device.

Strong intermolecular attractive forces exist between molecules tocreate surface tension. N. Vallespi i Salvado et al, “Surfactants inPressure Sensitive Adhesives”, Surface Coatings International, 4, 1999,pp. 181-185. These intermolecular forces create high surface tension inaqueous biological fluids such as blood, urine, and sputum. Incomparison, the surface energy of solid substrates is low. Thisdifferential between the surface tension of biological fluid andsubstrates commonly used to make in-vitro diagnostic devices needs to beovercome to achieve lateral flow and wicking.

Two approaches can be used to improve the flow of biological fluidsthrough a diagnostic device. One approach is to increase the surfaceenergy of the substrate (or membrane) with various surface treatments. Asecond approach is to reduce the surface tension of the biologicalfluid.

Adhesives are typically hydrophobic polymers with a surface energyranging from 30 to 40 dyne cm⁻¹. An approach to increase the flowproperties of in-vitro diagnostic devices is to increase the surfaceenergy of the hydrophobic adhesive coating. There are a number ofpatents that describe the synthesis and utility of hydrophilic polymersand adhesives.

For example, U.S. Pat. No. 3,686,355 describes a block copolymer of abase polymer with a second surface modifying additive. U.S. Pat. Nos.5,354,815 and 5,614,598 describe polymers having enhanced hydrophilicityand thermal regulated properties. In this area, a hydrophilicpolysiloxane anionic polymer is bonded to an aliphatic polyamide orpolyester polymer fiber to enhance the hydrophilic and thermalproperties of the textile. A number of U.S. and foreign patents aredirected to the use of hydrophilic polymers used to formulate pressuresensitive adhesives. See, for example, U.S. Pat. No. 5,508,313(hydrophilic pendant moieties on polymer backbone), U.S. Pat. No.5,660,178 (hydrophilic crosslinking agents), U.S. Pat. No. 6,121,508(lipophilic pressure sensitive adhesive with a surfactant for skincontact in biomedical electrodes), WO 00/56828 (use of hydrophilic estermonomers that are polymerized to produce a wet stick pressure sensitiveadhesive), EP 869979B (preparation of hydrophilic pressure sensitiveadhesive using polar monomers), U.S. Pat. No. 5,685,758 (hot meltadhesive with improved wicking for application to non-woven fabric), WO97/48779 (hydrophilic hot melt adhesive composition prepared by blendingadhesive components with a surfactant), and U.S. Pat. No. 6,040,048(water removable pressure sensitive adhesive containing hydropilicpendent groups).

Polymeric films have modified surface properties are well known andproduced by many distinct methods. See, for example, U.S. Pat. Nos.2,502,841 (gaseous chlorine); 2,829,070 (halogen gas); 3,142,582 (acidbath); 3,326,742 (halogenated organic amine); 3,561,995 (reactiveconditioning agent with metal ion); 3,843,617 (aqueous acidic solution);3,968,309 (surfactant-containing curable coating); 4,190,689 (titaniumdioxide treatment); 4,387,183 (grafting hydrophilic chains to polymersurface); 4,416,749 (irradiation and surface hydrolysis); 4,460,652(grafted hydrophilic polymer coating); 4,595,632 (hydroxy-fluorocarbongraft surface treatment); 4,666,452 (surface modified by hydrogensulfato groups); 5,273,812 (hydrophilic film of hydrophilic monomertogether with surface active agent); 5,280,084 (surface modificationwith carboxyl, carbonyl and hydroxyl groups followed by reaction withheterocyclic compound); 5,332,625 (crosslinked polymer surface);5,451,460 (coating of non-ionic, hydrophilic surfactant in binder);5,503,897 (irradiation and alkalization of polymer surface).

The present invention is directed to the selection of multifunctionalcoatings, adhesives and films and their use in in-vitro diagnosticdevices. Hydrophilic substrates or constructions can be hydrophilic heatseal coatings as well as pressure sensitive adhesive tapes. Pressuresensitive adhesive tapes facilitate device manufacturing and areintegral to device performance. The combination of a pressure-sensitiveor heat-sealable adhesive with hydrophilic properties to aid lateralflow and the wicking of biological fluids will prove beneficial todevice manufacturers. Benefits will include increased flexibility indevice design, increased wicking rates and consequently faster testresults. Increased wicking consistency and potentially reduced samplevolume are some of the advantages to be achieved through the use ofhydrophilic pressure sensitive adhesives and heat-sealable coatings.

In view of the above, the objects of the present invention are toprovide adhesive coatings or films with controllable hydrophilicity toincrease the surface energy of the fluid flow path to enhance the flowof biological fluids in in-vitro diagnostic devices, provide hydrophilicadhesives that bond components of the diagnostic device therebyfacilitating a more efficient manufacturing process for production ofthe device, increase the transfer rate of the sample fluid from an inletport to distal reagents and therefore reduce the time for analysis,enable smaller sample volumes by enabling more efficient transport offluid to a sensing reagent, and reduce risk of chemical interference byproviding a wicking surface that allows an increased separation betweenthe sampling port and the test reagents.

Hydrophilic coatings or films formulated by mixing surfactants with apolymer resin enhance the wicking of biological fluids into or throughan in-vitro diagnostic medical device. Polymer resins may be selectedfrom film forming polymers with a suitable glass transition temperatureto form a hydrophilic coating. Similar resins may be selected for heatsealable hydrophilic coatings. In addition, resins typically used aspressure sensitive adhesives may be formulated with surfactants toprovide a hydrophilic pressure sensitive adhesive. These constructionsare dual functional in that they may serve to bond the components of thediagnostic device together and also to create high energy surfaces whichreduce the surface tension of the biological fluid. The reduced surfacetension of the fluid allows rapid transfer of the fluid from an inletarea to a remote reagent area in an in-vitro diagnostic device. Therapid fluid spreading can reduce the time needed for analysis. Since asmaller sample volume is required due to effective fluid wicking, devicedesign flexibility is enhanced. This permits more efficientmanufacturing processing with the potential for reduced product cost.

Hydrophilic coatings, films and adhesives can also be employed which donot require the incorporation of the surfactant into the formulation toprovide the necessary hydrophilic properties. Examples of hydrophiliccoatings and adhesives include polymers that can be cross-linked usingdi-hydroxyl terminated polyethylene glycol or polypropylene glycolmonomers such as polyethylene glycol 600 supplied by Union CarbideCorporation. In addition, a vinyl terminated monomer with a hydrophilicmoiety such as an anionic group can be grafted onto a polymer backing toincrease the hydrophilic properties of the backing. One monomer that canbe used is sodium AMPS, which is the sodium salt of2-acrylamide-2-methyl-propanesulfonic acid, supplied by Lubrizol, whichcan be grafted onto the surface of a polymer by use of UV radiation.Such hydrophilic coatings can be used with and without the addition of asurfactant to provide a hydrophilic coating or adhesive.

Surface tension of a fluid is the energy parallel to the surface thatopposes extending the surface. Surface tension and surface energy areoften used interchangeably. Surface energy is the energy required to weta surface. To achieve optimum wicking, wetting and spreading, thesurface tension of a fluid is decreased and is less than the surfaceenergy, of the surface to be wetted. The wicking movement of abiological fluid through the channels of a diagnostic device occurs viacapillary flow. Capillary flow depends on cohesion forces between liquidmolecules and forces of adhesion between liquid and walls of channel.The Young/Laplace Equation states that fluids will rise in a channel orcolumn until the pressure differential between the weight of the fluidand the forces pushing it through channel are equal. Walter J. Moore,Physical Chemistry 3^(rd) edition, Prentice-Hall, 1962, p. 730.Δp=(2γ cos θ)/rwhere Δp is the pressure differential across the surface, γ is thesurface tension of the liquid, θ is the contact angle between the liquidand the walls of the channel and r is the radius of the cylinder. If thecapillary rise is h and ρ is the density of the liquid then the weightof the liquid in the column is πr²ghρ or the force per unit areabalancing the pressure difference is ghρ.

Therefore (2γ cos θ)/r=ghρ or h=2γ cos θ/gρ. For maximum flow throughmembranes (fluid wicking), the radius of the channel should be small,the contact angle θ should be small and γ the surface tension of thefluid should be large.

Wetting is the adhesion on contact between a liquid and solid. W. A.Zisman, “Influence of Constitution on Adhesion”, Handbook of Adhesives,2^(nd) edition, Van Nostrand Reinhold Co., 1977, p. 38. For maximumwetting, the surface tension of the liquid must be less than or equal tothe surface tension of the solid surface. This is the critical wettingtension of the solid. FIG. 3 illustrates surface wetting of a fluid on aflat smooth surface.

The theoretical explanation of this phenomenon can be described by theclassical model know as Young's Equation. T. Young, Philos. Trans. Roy.Soc. London, 95 (1805) p. 65.γ_(SV)=γ_(SL)+γ_(LV) cos θ  Eq.1

The diagram shown in FIG. 3, illustrates the relationship between thecontact angle θ and surface tension of liquid γ_(LV) and solid γ_(SV).W. A. Zisman, ibid, pp. 33-64. When the contact angle θ between liquidand solid is zero or so close to 0, the liquid will spread over thesolid.

The spontaneous process of wettability can also be derived from thedifferential between work of adhesion and cohesion by substitution ofDupre Equation below in Equation 2:W _(A) −W _(C)=γ_(SV)+γ_(LV)−γ_(SL)−2γ_(LV)=γ_(SV)−(γ_(LV)+γ_(SL))  Eq.2

This equation implies that spontaneous spreading will occur if the workrequired separating the liquid-solid interface is greater than liquidseparation itself. Therefore, Equation 2 can be further derived byintroducing the initial spreading coefficient S defined by Harkins (“ThePhysical Chemistry of Surface Films”, Reinhold, 1952) and shown inEquation 3 below:S=W _(A) −W _(C)=γ_(SV)−(γ_(LV)+γ_(SL))  Eq.3Since γ_(SL) is relatively small in comparison with γ_(LV), the initialspreading coefficient term becomes:S=γ _(SV)−γ_(LV)  Eq.4

Spreading is the movement of liquid across a solid surface. Contactangle is a measure of wettability. Spreading increases as the contactangle decreases until wetting is complete. Hence, the spreading willoccur spontaneously when S is greater than zero, which also indicatesthat the surface tension of the solid must be greater than that of theliquid, as shown in Equation 4. From the initial spreading coefficientequation showed above (Eq. 4), the wettability will occur either byincreasing surface tension of the solid or decreasing surface tension ofliquid.

Surface treatments can be used to increase the surface energy of a solidinclude both physical and chemical methods. Corona discharge, mechanicalabrasion, flame and plasma treatment are techniques used to increasesurface energy. P. H. Winfield et al, “The Use of Flame IonizationTechnology to Improve the Wettability and Adhesive Properties of Wood”,Int'l Journal of Adhesion and Adhesives, Vol. 21(2), 2001. Chemicalsurface treatments include cleaning, priming, coating and etching tochange the surface energy. Corona discharge treatment is the most widelyused technique for surface treatment of plastics. During the treatment,the plastic surfaces are heavily bombarded with oxygen radicals athigh-energy radiation levels. Consequently, the plastic surface eitherundergoes electret formation (J. M. Evans, J. Adhesion, 5 (1973) pp.1-7) or chemical structural changes (J. M. Evans, J. Adhesion, pp. 9-16;D. K. Owens, J. App. Polymer Science, 19 (1975), pp. 275-271 and3315-3326). Either proposal will improve the wettability of plastics.Another commonly used method is wet chemical treatment. This treatmentinvolves oxidizing the plastic surface through exposure to oxidizingacids such as a mixture of chromic acid and sulfuric acid. (D. Briggs etal, Journal Material Science, 11 (976))

Six commonly used industrial plastics were selected for study. Generalinformation for each plastic is listed in Table 1:

TABLE 1 General Information for Selected Plastics Plastic & AbbreviationProduct Name Manufacturer Polypropylene (PP) Amoco Amoco PolypropyleneChemical Company High Density Polyethylene Petrothene HD Quantum (HDPE)5003C Chemical Corporation Polycarbonate (PC) Cyrolon UVP PC CyroIndustries Polyethylene terephthalate (PET) Rynite Du Pont Poly methylmethacrylate (PMMA) Acrylite Cyro IndustriesAcrylonitrile-butadiene-styrene Cycolac GE Plastics terpolymer (ABS)GPX3700-1000

Each plastic was treated using two methods: 1) corona discharge and 2)chromic acid. The corona discharge treatment involved exposing thesurface of each plastic to an electric discharge of 10,000, to =50,000volts at a frequency of approximately 500 kilohertz for approximately 5seconds. The chromic acid treatment required the plastic surface to beflooded with chromic acid for 15 seconds then the acid was removed bywashing with distilled water then rinsing the surface with isopropanolthen wiped dry. The contact angle was measured immediately after dryingor corona treatment using the method described below. The contact anglewas measured on each plastic to quantitatively determine the effect ofeach treatment on the surface energy.

The data in FIG. 4 shows that water contact angles on treated plasticsdecrease indicating an increase in surface energy. Consequently, thewettability of biological fluids will also be enhanced as a result ofthese treatments. Both corona discharge and chromic acid treatments wereeffective in improving the wettability of the surfaces. Corona dischargewas most effective in increasing the surface energy of the polyolefinfilms (PP and HDPE) while chromic acid was more effective on plasticswith more reactive groups such as polycarbonate and polyester panels.The corona discharge treatment method could improve the water contactangle by orienting surface electrical charges or by introducing oxygenon the surface. Either mechanism will increase the polarity of theplastic and thereby increase its surface tension. Consequently, thecontact angle θ will be smaller due to reduced difference in surfacetension between the plastic γ_(SV) and the water γ_(LV). A disadvantageof corona discharge treatment is the instability of the treatment.Corona treated substrates should be coated soon after treated.

The use of surfactants to lower the surface tension of a fluid is wellknown. M. J. Rosen, Surfactant and Interfacial Phenomena John Wiley &Sons, New York, (1978); Th. F. Tadros, Surfactants, Academic Press, Inc.New York, (1984); A. C. Clark et al, “New and Improved WaterborneSystems”, Adhesives Age, September (1999), 33-40.

The effect of surfactants in coatings and adhesives has been studied todetermine their effect on wettability, fluid flow rate and adhesiveproperties. Each surfactant was formulated into a base adhesive atdifferent concentrations. The water contact angle was measured todetermine the effect of surfactant on reducing the surface tension ofthe water.

TABLE 2 Physical Properties of Selected Surfactants Charge Mol. ChemicalDescription Structure Types Wt. Sodium 2-Ethylhexyl Sulfate BranchedAnionic 232 Sodium Lauryl Sulfate Linear Anionic 288 Sodium NonylphenolEther Sulfate Aromatic Anionic 498 Nonylphenol Ethoxylate AromaticNonionic 704 Polyalkyeneoxide Modified Linear Nonionic 600Heptamethyltrisiloxane Siloxane

Hydrophilic coatings and heat-sealing and pressure sensitive adhesiveswere prepared. Dissolution of polymeric resins occurred in organicsolvents. Dissolution was followed by measurement of solution solids andviscosity over a period of several hours.

The surfactant was introduced into the liquid polymer mixture afterdissolution of the resin. Gentle agitation for several minutes wassufficient to achieve homogeneity. Hydrophilic pressure-sensitiveformulations were prepared by the introduction of a surfactant intoliquid acrylic adhesive solutions and emulsions followed by gentlymixing until dispersed or dissolved.

Hydrophilic films were prepared in the lab using coating apparatus. Thelab preparations were accomplished by use of coating bars that evenlyspread the liquid formulations on a film backing as shown in FIG. 5. Theliquid adhesive was first deposited as a pool onto a film backing, thenthe backing drawn through two stainless steel bars until the adhesivesolution spread across and down the film to produce an even coatingthickness. The thickness of the film was controlled the gap set betweenthe two coating bars. The cast films were dried for five to ten minutesin a Blue M Stabil Therm convection oven set at 105° C. The driedcoatings had an approximate thickness of 0.0005 to 0.001 inches, asmeasured with a Mitutoyo Absolute Digital Thickness Gage. Thehydrophilic adhesive coatings were protected with a film substrate oflow surface energy (release liner).

The hydrophilic coatings were tested for surface wetting usingde-ionized water. The sessile drop method was employed to measure thecontact angle liquid water makes with the surface of the hydrophilicthin film. A ramé hart contact angle goniometer was used.

A micropipette was used to draw deionized water from a beaker. Severaldrops of the liquid were dispensed back into the beaker to ensure abubble free liquid. The micropipette was then mounted onto thegoniometer.

An approximate 1″×1″ sample of hydrophilic film was place on thegoniometer stage with the hydrophilic surface towards the drop. The filmwas flattened then secured to the stage by placing a magnet or clamps oneach side of the film. Gloves were used when handling the film surfaceto avoid any oils or dirt from hands that could alter the surface of thefilm.

The micropipette was then lowered to just above the hydrophilic surface.A drop of water with a volume of approximately 2 μl was suspended on thetip and lowered towards the film until the water drop dispensed onto thesurface. The drop of water was allowed to spread across the surfaceuntil equilibrium was established (30 seconds). The microscope wasfocused to view the extreme left or right of the resulting drop (seeFIG. 6). The cross-line inside the scope was adjusted to tangency abovethe base of the drop to create a wedge of light bounded by the twocross-lines and the drop profile. The cross-line was slowly rotatedwhile adjusting the cross travel of the specimen stage assembly so thatthe wedge of light is gradually extinguished and the cross-line attainstangency with the drop profile at the base of the drop. The contactangle was read directly from the scope reticle at the six o'clockposition. The contact angle was recorded to the nearest degree on bothsides of the spread water drop.

Samples of the hydrophilic pressure sensitive and hydrophilic heat sealadhesive substrates were tested for peel adhesion to stainless steelpanels. Testing was performed on a MTS Alliance RT/1 mechanical testerequipped with a 25-lb load cell and hydraulic grips. The machine wasinterfaced with a Dell Optiplex GX1p computer system containing MTSTestWorks software package and Hewlett Packard 895c printer.

The hydrophilic pressure sensitive adhesive tapes were tested for peelstrength from 6″×6″ stainless steel panels using Adhesives Research ART1005, “Five Minute Peel”. The method is similar to ASTM D3330-83. Thetesting was carried out in a controlled temperature (70° F.) andhumidity (50% RH) environment. Prior to testing, the stainless steelpanels were cleaned with high purity urethane grade 2-butanone. The tapesamples were cut to 1″×10″, then laminated (two passes) to the stainlesssteel panel using a 4.5-lb, 80 durometer hardness roller. Peel testingwas initiated after a five-minute dwell, by attachment of stainlesssteel plate to the bottom set of grips and overhanging, unbound portionof tape to the top set of grips. The tape was pulled away from thestainless steel plate at a rate of 12 inches/minute and at an angle of180-degrees. The load and displacement were observed to increase to amaximum over the first 1-inch of the test then remain constant until thetest was complete. The peel strength was calculated from the quotient ofaverage load (oz) between one and five-inch displacement on the panel,and the sample width (in).

Hydrophilic heat seal coatings were tested for adhesion using ASTMD1876-95. The testing was carried out at 70° F. and 50% relativehumidity. Adhesion was tested after lamination to a cleaned 7-milpolyester film. The samples were heat laminated on a Wabash press byexposure for 2 seconds at 100° C. and at a pressure of 30-40 psi. Thesamples were aged four days at 70° F. and 50% R. H. Peel testing wasconducted as described above and found to be acceptable.

The effect of hydrophilic coatings and adhesives on the flow rate ofdistilled water in a microfluidic channel was investigated. Following ascreening of the effect of different types of surfactants on contactangle, the most effective surfactants were formulated into adhesivetapes that were used as a cover for a microfluidic device as shown inFIG. 7.

The microfluidic channel was molded in a device made from polystyrene.The channel had a length of 20 cm with a depth of 10 microns and a widthof 30 microns. The hydrophilic tape was used to close the channel tocreate the microfluidic device. Distilled water was placed in one of theterminal wells and the time for the water to flow through the channelwas measured.

Chemical surface analysis of the hydrophilic coatings was performedusing infrared spectroscopy via attenuated total reflectance (ATR). Thespectra were recorded using a Pike Miracle ATR single bounce sample portunit using a ZnSe crystal. Film samples were compressed onto the crystalusing the compression arm at full contact pressure. Infrared spectrawere collected using a MIDAC M1300 Series FT-IR bench with amercury-cadmium-telliuride (MCT) nitrogen cooled detector. Absorbancespectra were collected from 30 scans per sample from 4000 cm⁻¹ to 600cm⁻¹ at 2 cm⁻¹ resolution at a gain of 1X. The FTIR bench was interfacedwith YKE Microsystem computer and analyzed using Grams 32 softwarepackage.

The surface topography of the hydrophilic coatings was observed usingatomic force microscopy. The instrument used was a Digital InstrumentsNanoscope IIIa Multimode instrument. Hydrophilic tapes were mounted onto1-cm diameter magnetic stubs and imaged in the tapping mode. Using thismode, the AFM cantilever is oscillated at its resonant frequency.Contact between the oscillating tip and the tape surface causes adecrease in the measured amplitude of oscillation. Since the contact ismade at the largest displacement from the cantilever equilibriumposition, little energy is transferred to the sample and minimaldeformation of the sample occurs. Images were obtained by rasterscanning the sample surface under the tip and recording the z motion ofthe sample necessary to maintain constant amplitude during the scan.This mode of imaging has several advantages over direct contact modeimaging. Lateral forces that are prevalent during contact mode scans areeliminated. Additionally, this tapping mode provides a non-destructivemethod for the imaging of soft samples. Importantly, phase imagesobtained using the tapping mode can give additional informationconcerning the mechanical and adhesive properties of the sample surface.A. Doring et al, “Atomic Force Microscopy: Micro- and Nano-Mapping ofAdhesion, Tack and Viscosity”, 23^(rd) Annual Technical Seminar:Pressure Sensitive Adhesive Tapes for the New Millennium, May, 2000, pp.213-222.

All samples were initially scanned in air. The hydrophilic coating HY-10was then rinsed with de-ionized water for 10 seconds before being wipeddry with a paper tissue. The sample was left to dry overnight and imagedthe next morning.

Various surfactants as shown in Table 2 were formulated into an emulsionpressure sensitive adhesive. FIG. 8 shows that most test samples exhibita similar trend of decreasing contact angle with increasing surfactantconcentration. Sodium nonylphenol ether sulfate exhibited the mosteffective surface tension reduction of water at all three surfactantconcentrations used in this study. The nonionic surfactant, nonylphenolethoxylate, exhibited little effect on the contact angle of de-ionizedwater. This may be due to its higher molecular weight and the lowerwater affinity of the hydrophilic group compared to anionic typesurfactants. In addition, the nonylphenol group enhances its absorptiononto the polymer surface. Polyalkyeneoxide modifiedheptamethyltrisiloxane (PMHS) (SILWET L77 from Union Carbide), also anon-ionic surfactant, reduced the water contact angle of the adhesivesurface compared with the nonylphenol ethoxylate. PMHS has a siloxanepolymer backbone instead of a hydrocarbon backbone, which accounts forits lower surface energy. In addition, PMHS also has a lower molecularweight than nonylphenol ethoxylate which enhances its mobility withinthe adhesive matrix. PMHS can be formulated into a solvent-basedpressure sensitivd adhesive in amounts of up to about 20% by wt. toincrease the hydrophilic prope'rties of the adhesive.

Of the surfactants evaluated, sodium nonylphenol ether sulfate had thehighest molecular weight of the anionic surfactant used. It is believedthat the lower molecular weight anionic surfactants have bettersolubility into the adhesive matrix so that the concentration of thesurfactant at the water/adhesive interface is less. The linear structureof sodium lauryl sulfate may improve its solubility into the adhesive sothat its effect on the adhesive surface is less than that of sodium2-ethylhexyl sulfate.

The wetting of the surface of HY-5 and HY-10 which are two hydrophilicheat seal adhesives was investigated by measuring the spreading ofwater. These hydrophilic heat seal adhesives were formulated usingpolyester resins and the anionic surfactants, sodium nonylphenol ethersulfate and sodium dioctylsulfo succinate, respectively. FIG. 9 is agraph that describes the spreading behavior of water on the surface ofHY-5 and HY-10 thin film coatings. Water was dropped onto the surface ofthe adhesives and the contact angle was measured as a function of time.Initially there is rapid spreading of the drop as it contacts thesurface of the film. The contact angle decreases quickly to less than 10degrees. Equilibrium is established within thirty seconds to one minute.This spreading behavior is typical of the hydrophilic coatings,heat-seal adhesives, and pressure sensitive adhesives.

FIG. 10 shows the effect of the surfactant concentration on the surfacewettability of the dried films prepared using different polymericresins. Polyamide, ethylene vinyl acetate, and polyester resins wereformulated with sodium dioctylsulfo succinate. The resins studiedincluded films of polyamide, ethylene vinyl acetate, and polyesterchemistries. When no surfactant is present in the coatings the contactangle is high since the polymeric resins are hydrophobic. By increasingthe surfactant concentration the surface becomes more hydrophilic andlower water contact angles are observed indicating significant surfacewetting. At very high surfactant concentrations the wetting effect canbe enhanced or attenuated depending on the surfactant and itscompatibility with the polymer matrix.

FIG. 11 shows the effect of surfactant concentration on the rate ofwater flow in a covered microfluidic device (corresponding to device ofFIG. 7). In this experiment, a hydrophilic pressure sensitive adhesivewas formulated using concentrations of sodium nonylphenol ether sulfateranging from 0 to 6 percent. When there was no surfactant added to theadhesive, water did not flow through the channel. With increasingconcentration of surfactant the rate of water flow through themicrochannels increased while the contact angle decreased.

The increased flow rate of water can be attributed to the reduction ofwater surface tension. The principle that could be used to explain thisphenomenon is capillary rise as shown in FIG. 2 which documents therelationship between the surface tension and the contact angle. Theheight of the water in the capillary is determined by a factor of twotimes the product of liquid surface γ_(LV) and cos θ regardless ofliquid density and gravitation force. As a result, the water willadvance further when the surface tension of water is close to thesurface tension of the capillary material that is now determined by thehydrophilic adhesive cover. At high surfactant concentration (greaterthan 4% in FIG. 11), the rate of flow levels off since the concentrationof surfactant exceeds the critical micelle concentration. Additionalsurfactant on the surface of the adhesive does not reduce the surfacetension of the fluid and may become autophobic. W. A. Zisman, “Influenceof Constitution on Adhesion”, Handbook of Adhesives, 2^(nd) edition,1977, p. 46.

Atomic force microscopy (AFM) was used to visualize the topography of ahydrophilic coatings. The AFM images of coatings containing 0%, 1%, 5%and 10% surfactant were obtained. The images show enrichment of the filmsurface at the film/air interface with increasing amount of surfactantintroduced to the adhesive formula. The AFM image of the coatingcontaining no surfactant shows a relatively smooth, flat surface.

Transformation is observed when 1% or less surfactant has beenincorporated into the adhesive coating, where raised surface featuresare observed on the film surface. Increased surface topography isobserved at 5% surfactant while at 10% surfactant the surface appears tobe smoother due to saturation of the surface.

Infrared spectra of the coatings confirm the increase in surfactantconcentration on the surface. The prominent peak at 2958 cm⁻¹ in the ATRis assigned to the C—H stretch of a CH₃ group on the surfactant in thehydrophilic adhesive and is used to monitor surfactant accumulation onthe surface. A plot of absorbance of the C—H stretch as a function ofconcentration of surfactant at 0%, 1.0%, 5.0% and 10% shows a flatteningresulting from the surface saturation by the surfactant

Hydrophilic coatings, hydrophilic pressure-sensitive and heat-sealableadhesives may be used in a variety of in-vitro diagnostic devices,including capillary flow, lateral flow, microfluidic, microtiter platesand electrophoretic devices.

The following are examples of various embodiments of the presentinvention:

Example 1 Hydrophilic Coating

A polyester resin with a high glass transition temperature commerciallyavailable as Vitel 2200 BA from Bostik Chemical Company is dissolved ina solvent of methyl ethyl ketone and toluene (7:3 weight ratio). Acommercial surfactant such as Rhodapex CO-436 available from Rhodia Inc.is dissolved in the resin solution to provide a surfactant solids toresin ratio of between 3:97 to 6:94. A hydrophilic coating is formed byspreading the resin/surfactant solution onto a polymer film and allowingthe solvent to evaporate. Wetting the dried film surface with distilledwater causes spreading of water on the surface. The contact angle of thewater on the surface ranges from 5 to 10 degrees.

Example 2 Hydrophilic Heat Sealable Coating

A similar formulation and coating as in Example 1 is prepared using apolyester resin with a lower glass transition temperature such as Vitel3200 through 3500 series resins with a glass transition temperaturebetween −15° C. to +15° C. One example is Vitel 3300B which has a Tg of+11° C. A heat sealable hydrophilic coating is formed by coating theformulation of resin and surfactant onto a surface such as a polymericfilm and allowing the solvent to evaporate. The contact angle of thehydrophilic coating is similar to those of Example 1 (5 to 10 degrees).Other resins such as ethylene vinyl acetate and polyamide polymers maybe used as heat sealable formulations.

Examples 3-8 Hydrophilic Pressure Sensitive Coatings Aqueous Based

A hydrophilic pressure sensitive coating is prepared by formulating anemulsion based resin such as Aroset 3500 available from AshlandSpecialty Chemical Company (division of Ashland, Inc.), with asurfactant such as Rhodapex CO-433 available from Rhodia, Inc. Theformulation was coated onto a hydrophobic polymer film such as 5 milpolyester film available from DuPont Teijin Films. After coating anddrying the formulation, the contact angle was measured. The followingtable illustrates the effect of surfactant concentration on the contactangle which is related to the surface energy of the hydrophilicadhesive. The adhesive 180° peel force can be modified through additionof additives such as tackifiers.

Acrylic Resin (%) Rhodapex CO-433 Contact Angle 180° Peel AqueousSolvent (%) (Degrees) (oz/inch) 99 1 95 98 2 32 68 97 3 29 96 4 15 6 955 13 94 6 11 7

Examples 9-12 Hydrophilic Pressure Sensitive Coatings Solvent Based

Similar to Examples 3-8, hydrophilic pressure sensitive adhesivecoatings are formulated using solvent based adhesives and surfactants.An acrylic resin adhesive in the organic solvent ethyl acetate wasformulated with various concentrations of Rhodapex CO-433. After coatingonto a polyester film and drying, the contact angle of the coating wasmeasured. The following table shows the effect of surfactantconcentration on the contact angle and 180° peel force.

Acrylic Resin (%) Rhodapex CO-433 Contact Angle Ethyl Acetate Solvent(%) (Degrees) 97 3 33 94 6 17 91 9 16 88 12 15

In addition to the concentration of the surfactant, the surface energyof the hydrophilic coating can be controlled by the selection of surfaceactive agent. The selection of surface active agent is based on factorssuch as molecular weight, linear vs. branched structure, ionic vs.non-ionic and the type of ionic moiety present, aromatic vs. aliphaticstructure, etc. These chemical structure properties can be used tocontrol the hydrophilic characteristics and surface energy of thecoating. The following table shows the effect on contact angle of 2percent surfactant in Aroset 3500 coatings by the selection ofsurfactant structure. The following table shows the effect of surfactantcharacteristics on coating wettability:

Surfactant/ Contact Angle Ionic Charge Molecular Wt Structure Sodium 2-anionic 232 branched Ethylhexyl sulfate 41° Sodium octyl anionic 232linear Sulfate 19° Sodium lauryl anionic 288 linear Sulfate 20° Sodiumanionic 382 aromatic Nonylphenol sulfate 32° Nonylphenol nonionic 820aromatic Ethoxylate 105°

A novel feature of using a hydrophilic coating formulated with RhodapexCO-436 (the ammonium salt of sulfated nonylphenol ethoxylate) is theability to pattern the surface energy of a uniform coating using radiantenergy. When thermal energy is applied to the coating in a pattern suchas stripes, circles or any other configuration, the surface energy inthe area of applied energy is reduced. It is believed that ammonia gasis evolved due to the thermal energy leaving the sulfonic acid ofnonylphenol ethoxylate remaining. The hydrophilicity of the coatingdecreases and consequently becomes water resistant. Radiant energysources such as lasers and electron beam may also be employed to causethe evolution of a labile cation to customize the physical character ofthe coating. This may be used with advantage in the production ofin-vitro diagnostic devices, such as by the application of thermalenergy to the surface to produce a parallel, laterally-oriented, stripedpattern of alternating hydrophilic/hydrophobic areas. The presence ofthe hydrophobic areas may be employed with advantage to slow the wickingof the material to be tested as it travels from a hydrophilic region toa hydrophobic region, whereby additional time for reaction between theanalyte and the reagent results. Fluid wicking through the device may beretarded over areas of lower surface energy to permit time for reactionor complex formation by use of a single coating. This may be employed toavoid too rapid fluid wicking which may be detrimental if the reactiontime is insufficient. Of course, a series of reaction zones of variousshapes and configurations can be created on a single film.

The present invention may be employed with advantage in a variety ofin-vitro diagnostic devices, both of the lateral flow and of thecapillary flow type, with devices of the lateral flow rate type of FIGS.12-17. In one embodiment of a lateral flow device of the presentinvention as depicted in FIG. 15, the device comprises a housing cover1, means (port) 3 in the housing to introduce a sample to be assayedinto the device, means 5 (absorbent pad) for fluid collection, and abacking strip 7 having spaced apart first and second ends. The means forsample fluid collection is adhered to the backing at a first end of thebacking strip, the means to introduce the sample is adhered to thebacking at the second end of the backing strip. A microporous or porousmembrane 9 is optionally placed between the first and second ends toprovide an avenue for travel of the sample between the first and secondends as well as to provide a matrix for any reagent material that may bepresent for contact with the fluid sample, during which time the samplecontacts the reagent with which reaction or contact is to occur.

Advantageously, in accordance with the present invention, the surface ofthe backing strip between the first and second ends is hydrophilic incharacter. The backing strip 7 may be, e.g., heat-sealable or exhibitpressure sensitive adhesive properties. If the backing strip 7 exhibitspressure sensitise adhesive properties, the hydrophilic character of thematerial serves to avoid reducing the effectiveness of any membrane 9attached to the backing strip in the event that migration of theadhesive into the membrane occurs.

By way of further advantage, due to the hydrophilic character of thebacking strip, it may be possible to avoid use of the membrane 9,instead relying solely on the hydrophilic character of the backing stripitself to wick the sample from the sample introduction point to thesample collection point. In such an embodiment, the reagent with whichthe sample must contact or react with will either be applied directly tothe backing strip for contact with the sample, or be introduced to thesurface of the backing strip from a reservoir attached to the backingstrip in a conventional manner.

Port 11 may be employed to provide access for another material such as abuffer to be applied to absorbent pad 13. The sample once added to port3 contacts absorbent pad 15. The assembly of the backing strip andassociated attached components may be positioned within a bottom portion17 of the housing. The housing cover 1 includes view port 20 for viewingthe visual result of the reaction between the sample and the reagentpresent in the device.

FIGS. 12 and 13 depict a lateral flow test strip according to thepresent invention. The test strip includes sample absorbent pad 19,membrane 21 and sample collection pad 23. Backing strip 25 includes ahydrophilic surface 27 which may be heat-sealable or pressure sensitivein nature in accordance with the present invention. Areas 29 on themembrane 21 contain reagents for reaction with the sample.Alternatively, the membrane may be omitted and its function served bythe hydrophilic surface of the backing strip 25. In such an embodiment,the areas 29 may still contain reagents for reaction with the testsample, and areas 29 of the backing strip may also be made morehydrophobic (or less hydrophilic) than the remaining surface of thebacking strip. The presence of such areas will serve to slow the rate ofpassage of the sample across the backing strip to maximize time ofcontact with the reagents in areas 29.

Another embodiment of the device of the present invention is depicted inFIG. 14. The device of FIG. 14 includes covers 31,33 for the respectiveends of the device, which include sample pad 37 and collection pad 35,with test zones 41 being intermediate the ends of the device on backingstrip 39 having a hydrophilic surface 43. As discussed above, test zones41 may be positioned on portions of the backing strip which have beenrendered less hydrophilic (or more hydrophobic) than the remainingportion of the backing strip.

Various modifications can be undertaken with advantage in such anembodiment. As discussed above, selective areas ofhydrophilic/hydrophobic surface character can be provided on the surfaceof the backing material to modify the flow characteristics of the fluidsample, either by directing the sample longitudinally along the backingstrip toward the fluid collection point, or by causing the fluid sampleto contact adjacent hydrophilic/hydrophobic areas to slow the flow rateof the fluid sample along the backing strip. In such an instance, forexample, the reagent may be placed on the hydrophobic portion where thewicking of the fluid sample would be slower to permit a longer contacttime with between the fluid sample and the reagent. In terms of thisdiscussion, the term hydrophobic is not intended to mean that theportion of the backing would be entirely hydrophobic, but could alsomean that that the area is more hydrophobic than the adjacenthydrophilic portion of the backing strip (i.e., both portions would havevarying degrees of hydrophilicity so that the wicking of the fluidsample would still be encouraged to travel from the sample inlet to thesample collection area).

Accordingly, in the context of FIGS. 12-15, the surface of the backingfilm (e.g. a polyester film as in FIG. 1) could be rendered hydrophilicby any of the methods discussed above, and employed as a heat-sealablelayer for bonding to the absorbant pad and the sample pad/conjugate pad.Optionally, a c membrane could also be bonded to the heat-sealablehydrophilic backing strip. Alternatively, the use of the membrane can beavoided and the reagents applied directly to the hydrophilic surface ofthe backing strip and the sample and reagent caused to wick directlyacross the surface of the backing strip toward the absorbent pad.

As discussed above, in an embodiment where the backing strip comprises ahydrophilic pressure sensitive adhesive layer, the membrane can still beused with advantage due to the hydrophilic character of the adhesivewithout fear of diminishment of the ability of the membrane to functiondue to migration of the adhesive. However, it is still possible to avoidthe use of the membrane, with the hydrophilic adhesive layer serving asthe transport medium for the sample from the sample pad to the absorbentpad. Any reagents desired to be contacted with the sample may be applieddirectly to the surface of the hydrophilic adhesive layer. The adhesivecharacter of the backing strip can also be employed with advantage tobond the respective sample/conjugate/absorbent pads to the backingstrip. This facilitates the manufacture of the device. Such a devicewould typically be contained in a suitable housing that generallyincludes a viewing window to determine the extent of the reaction of thesample and the reagent (e.g., to determine extent of reaction due tocolor formation or the intensity of the color formed).

In the context of a microfluidic diagnostic device which employscapillary transport of the fluid sample during the analysis procedure,such devices typically include microfluidic channels molded in asuitable polymeric substrate (see FIGS. 7 and 16). Microfluidic devicesgenerally refers to a device having one or more fluid channels,passages, chambers or conduits which have at least one internalcross-sectional dimension (width or depth) of between 0.1 um and 500 mmwithin which a fluid sample passes from an inlet port to a detectionzone.

The microfluidic diagnostic device is generally comprised of asubstantially planar base portion having one or more microfluidicchannels, passages, chambers or conduits therein. A variety of materialsmay comprise the base portion, including polymeric materials such aspolymethylmethacrylate, polycarbonate, polytetrafluoroethylene,polyinylchloride, polydimethylsiloxane, polysulfone, and silica-basedsubstrates such as glass, quartz, silicon and polysilicon, as well asother conventionally-employed substrate materials.

Such substrates are manufactured by conventional means, such as byinjection molding, embossing or stamping, etc. The microfluidic passagesor channels may be fabricated into the base portion by conventionalmicrofabrication techniques known to those skilled in the art, includingbut not limited to photolithography, wet chemical etching, laserablation, air abrasion techniques, injection molding, embossing, andother techniques. The base material is selected on the basis ofcompatibility with the desired method of manufacture as well as forcompatibility with the anticipated exposure to materials and conditions,including extremes of pH, temperature, salt concentration, and theapplication of electric fields. The base material may also be selectedfor optional properties including clarity and spectral characteristics.

An enclosure surface or cover is placed over the top portion of the basesubstrate to enclose and otherwise seal the microfluidic passages orchannels. In the context of the present invention, the channels orpassages are covered with a substrate according to the present inventionthe surface of which is hydrophilic which covers the passages orchannels in the base substrate. The fact that the surface of thecovering substrate is hydrophilic in nature enhances the flow of theliquid through the microfluidic passages and channels. As discussedabove, the hydrophilic covering substrate can comprise a variety oftypes of materials having hydrophilic character, such as a hydrophilicpressure sensitive adhesive layer, a hydrophilic heat-sealable layer, ahydrophilic surface-treated layer, etc. Hydrophilic pressure sensitiveadhesives can be bonded to the upper portion of the base substrate incovering/sealing relation to the microfluidic passages/channels byapplication of pressure. Hydrophilic heat-sealable layers can be bondedto the upper portion of the base substrate in covering/sealing relationto the microfluidic passages/channels by application of pressure andheat, with the temperatures employed being sufficient to cause bondingof the covering layer without adversely affecting the physical structureof the base material. Other means of bonding the covering material tothe base substrate can be employed such as acoustic welding techniques,UV curable adhesives, etc.

Such devices typically include optical detector means positionedadjacent to a detector window whereby the detector senses the presenceor absence of an optical characteristic from within the microfluidicpassage or channel resulting from flow of the liquid sample through thepassage or sample. The optical detector may comprise any of a variety ofdetector means such as fluorescent, colorimetric or video detectionsystems, which include an excitation light source (laser or LED), etc. Avariety of optically detectable labels can be employed to provide anoptically detectable characteristic such as colored labels, colloidlabels, fluorescent labels, spectral characteristics andchemiluminescent labels.

As discussed above, an alternative to otherwise having to ensure thatthe channels possess sufficient hydrophilicity to cause the fluid sampleto travel along the capillary tube, the top portion of the channel iscovered with a hydrophilic material in accordance with the presentinvention. That is, a heat-sealable polymeric film having hydrophilicsurface characteristics may be applied over the open cavity of thechannel to both enclose the channel and provide the necessaryhydrophilic character so that the fluid sample will be caused to wet thechannel. As an alternative, the polymeric film may include a pressuresensitive adhesive coating which is also hydrophilic in character toprovide the necessary hydrophilicity to cause the fluid sample to wetthe channel. The use of such materials in the construction of themicrofluidic diagnostic device also serves to simplify the manufacturingof the device. In the context of the present invention, the entirefacing surface of the covering layer need not be hydrophilic; instead,only that portion of the covering layer that serves to enclose themicrofluidic channels or passages is required to be hydrophilic. Ofcourse, as is the case with lateral flow devices, certain portions ofthe covering layer that enclose the microfluidic channels or passagesmay be rendered less hydrophilic than other portions to modify the flowrate of the fluid sample.

A typical microfluidic device which has been prepared in accordance withthe present invention is depicted at FIGS. 16 and 17. The device of FIG.16 includes base portion 45, recess 47 in the top of the base 45, openmicrofluidic channels 49, fluid reservoirs 51 and viewing window 53. Inthe device of FIG. 16, the microfluidic channels 49 are uncovered inorder to depict the interior of the device. In the cross-sectional viewof the device of FIG. 16 (at FIG. 17), base portion 45 includesmicrofluidic channel 49 which is shown to be enclosed by cover portion55. Cover portion 55 includes a facing hydrophilic surface 57 wherebythe fluid sample which enters the microfluidic channel 49 will contactthe facing hydrophilic surface and cause the sample to be transportedalong the length of the channel. The facing surface 57 of the cover 55may be rendered hydrophilic by various means in accordance with thepresent invention, such as by the presence of a hydrophilic pressuresensitive adhesive, by the rendering of the surface of the cover itselfhydrophilic by suitable means such as by mechanical or chemicaltreatment, etc. For example, cover 55 may be heat-sealed or adhesivelyattached to the interior portion of the base 45.

By way of an alternative embodiment depicted in FIGS. 18 and 19, themicrofluidic in-vitro diagnostic device may be comprised of opposingbase layers 69, 75 separated by an adhesive spacer layer 71. While onlya single base layer is shown in FIG. 18 so as to depict the fluidchannels 73, both base layers are shown in FIG. 19. The spacer layer 71may have fluid channels 73 provided therein within which a fluid to beassayed passes from a reservoir to a collection point. At least aportion of the surfaces of the base layers 69, 75 and the spacer layerwhich define the boundaries of the fluid channels are hydrophilic incharacter. The requisite hydrophilic character may be provided inseveral ways. For instance, one or both of the base layers 69, 75 or theadhesive layer may be hydrophilic or rendered hydrophilic by any of themethods described herein. For example, the respective layers may becomprised of a polymeric material which is inherently hydrophilic,rendered hydrophilic as a result of a compounding step, or surfacetreated to provide the necessary hydrophilicity. The spacer layer 71preferably is an adhesive layer which is bonded to the opposing baselayers, either as a result of pressure sensitive adhesive properties ofthe spacer layer or as a result of being heat-sealed to each of the baselayers. If pressure sensitive, the spacer layer may be used in the formof a transfer film or as a double face construction. As discussed above,if the base layers are not hydrophilic in character, the spacer layerwould possess the requisite hydrophilic character to assist wetting ofthe fluid channel by the fluid sample. The fluid channels 73 in thespacer layer may be die-cut into the spacer layer or provided by anyother means effective to provide a spacer layer with the requisite fluidchannels. One advantage of such a construction is that the micro-fluidicdevice may be constructed easily without the need to mold the fluidchannels into the base layers as in the embodiment of FIG. 16.

Microplates of the present invention include various embodiments such asmicrowell-containing microplates as shown in FIGS. 20 and 21. As shownin the Figures, the microplate includes base portion 61 within which areformed a multitude of microwells 63. The microwells 63 may be of anysuitable configuration, such as hexagonal or cylindrical as depicted.FIG. 20 depicts the presence of a cover plate or sheet 65 on the top ofthe base portion 61 to seal the microwells. The cover plate or sheet maycomprise a heat-sealable film or may have pressure sensitive properties.As depicted in FIG. 20, a suitable material such as a lyophilizedsubstrate, etc. may, as desired, be attached to the inner surface of thecover plate or sheet in the event that the inner surface of the plate orsheet exhibits pressure sensitive adhesive properties, or by use ofother adhesive means. In the context of the present invention, the coverplate or sheet, at least on the inner surface thereof which covers themicrowells, will exhibit hydrophilic properties. Such properties can beprovided by use of a pressure sensitive adhesive which is renderedhydrophilic in the manner taught above, or by use of a heat sealablefilm which is similarly possesses hydrophilic properties also in themanner taught above.

An alternative microplate embodiment is shown in FIGS. 22 and 23 whichcomprises an open well microplate having a base portion 77 containing aplurality of microholes 79 cut or molded therein and passing completelythrough the base portion 77. The base portion 77 would be provided withfacing cover plates or layers in order to seal the respective microholes79 so that the respective liquid samples may be placed therein. Eitheror both of the base portion or the cover portions (not shown) adjacentthe holes would be rendered hydrophilic in character. The coveringplates or layers may be attached to the base plate by suitable adhesivemeans such as pressure sensitive adhesive or heat sealable adhesiveproperties of the cover plates or layers.

The present invention may employ a polymeric film which has been surfacemodified to exhibit hydrophilic properties. Polymers which can bemodified in this manner are well known in the art. Exemplary of suchpolymers are the following polymers: polyolefins, including but notlimited to polyethylene, polystyrene, polyvinyl chloride, polyvinylacetate, polyvinylidene chloride, polyacrylic acid, polymethacrylicacid, polymethyl methacrylate, polyethyl acrylate, polyacrylamide,polyacrylonitrile, polypropylene, poly(1-butene), poly(2-butene),poly(1-pentene), poly(2-pentene), poly(3-methyl-1-pentene),poly(4-methyl-1-pentene), 1,2-poly-1,3-butadiene,1,4-poly-1,3-butadiene, polyisoprene, polychloroprene, ethylene-vinylacetate copolymer, polycarbonate, ethylene-isobutyl acrylate copolymer,as well as random or block copolymers of two or more polyolefins or apolyolefin and a non-olefin. Similarly, blends of two or more polymersmay also be employed, as long as the polymer produced is hydrophobic incharacter.

The polymer may also comprise a polyester such as polyethyleneterephthalate, polyethylene isophthalate-terephthalate, copolymers ofpoly-(1,4-cyclohexane dimethylene)terephthalate, poly(1,4-cyclohexanedimethylene) isophthalate, and isophthalate-terephthalate copolymers;poly(1,4-phenylene) terephthalate and isophthalate and copolymers;poly(1,4-phenylene)-4,4′ diphenyl dicarboxylate; polyesters derived fromaliphatic dibasic acids, such as maleic, adipic and sebacic acids andpolyhydroxy compounds such as polyethylene glycol, neopentyl glycol,butylene glycol, glycerol, pentaerythritol, and cellulose. Preferably,the film-forming polymers used in the present invention exhibit a Tg orTc sufficient to permit the polymer to be film-forming as well as toenable the resulting polymer film to be heat sealable at a sufficientlylow temperature (e.g., in the range of from 70 to 100° C.).

As discussed above, a variety of surfactants may be admixed with thepolymer to render the surface of the polymer hydrophilic. Surfactantswhich are suitable for use in the present invention include anysurfactant which effectively imparts hydrophilic surface properties tothe hydrophobic polymer film. While the identity of such surfactants isnot critical to the practice of the present invention, anionicsurfactants are preferred. However, exemplary of such surfactants(without limitation) are ammonium salts or sodium salts of alkyl phenoxy(polyethylene oxy)ethanol, ammonium perfluoroalkyl sulfonates, etc.Exemplary surfactants preferably include one or more hydroxyl,carboxylic acid, sulfonic acid, and amine functionalities. A detaileddiscussion of surfactants resides in Kirk-Othmer, Encyclopedia ofChemical Technologies, 2^(nd) Edition, Vol. 19, pages 512-564, hereinincorporated by reference.

The above embodiment of the present invention may be practiced asfollows in order to result in the formation of a hydrophobic polymerfilm having hydrophilic surface properties. Initially, a hydrophobicfilm-forming polymer is admixed with a suitable solvent to form asolvated solution of the polymer in the solvent. The polymer is admixedand dissolved in the solvent under conditions which permit the polymerto be so dissolved. Such conditions may include, for example,temperatures within the range of from 20 to 30° C., although highertemperatures may also be employed depending upon the polymer and solventemployed. The solvent which is employed is dependent upon the selectionof the particular polymer. Exemplary solvents from which such selectionmay be made include but are not limited to toluene, methyl ethyl ketone,xylene, ethyl acetate, tetrahydrofuran, methylene chloride, n-heptane,n-butylacetate, acetone, cellosolve acetate, methyl cellosolve,n-butanol, isopropanol, n-propanol, and ethanol.

Once so formed, the solution of the polymer and the polymer solvent isadmixed with a suitable surfactant which is soluble within the solutionof the polymer and the polymer solvent. The surfactant is admixed in anamount of, for example, up to about 10% by weight, based on the totalweight of the polymer and surfactant. Preferably, the surfactant isadmixed with the polymer in an amount in the range of from about 3 to 6%by weight.

Once the mixture of polymer, solvent and surfactant is formed, themixture is cast or otherwise caused to be formed into a film. Thesolvent contained in the thus-formed film is then caused to be removedfrom the film by the application of heat or other means (such as byreduced pressure). The film which then results is comprised of ahydrophobic polymer which exhibits desirable homogeneous hydrophilicsurface properties. For example, an elevated temperature within therange of about 80° C. to 120° C. may be employed depending upon thevaporization temperature of the solvent. It is not advisable to employ atemperature much in excess of the vaporization temperature of thesolvent in order to avoid loss of homogeneity of the hydrophilic surfaceproperties exhibited by the resulting film.

It is frequently a disadvantage in any determination by means offluorescent detection that “background” fluorescence occurs which mayaffect the accuracy of the desired fluorescent detection. It haspreviously been proposed to employ “low background” assay platforms andwell plates for use in fluorescent detection methods to minimize thedegree of background fluorescence during the assay. See U.S. Pat. Nos.5,910,287 and 6,171,780 in this regard. These patents teach the use ofpolymers having low fluorescence and high transmittance such ascycloolefins in the formation of the bottom portion of the wells in amulti-well assay platform.

It would be desirable, however, to also employ a low fluorescent sealingor cover layer either alone or in conjunction with a low fluorescentassay platform or multi-well plates to further reduce the possibility ofundesirable background fluorescence during the assay by fluorescentdetection.

Fluorescence is defined as “radiative transition from the lowest excitedsinglet state (S₁) to the ground state (S_(o)) (Electronic Properties ofPolymers, ed. J. Mort et al, p. 177, 1982). Materials for applicationswhere no or minimal fluorescence is preferred (such as microfluidicdevices) typically have high excitation energy potential. In polymericmaterials, the base monomer preferably has a high ionization potentialand low electron affinity. High energy is required to excite themolecules from a ground state to an excited state. Low fluorescentcompounds do not easily accept charge transfer from other compounds orexcited states. Similarly, molecules that are easily polarized by thedelocatization of an electron should be avoided. Aromatic compounds andcompounds with a conjugated pi electron structure may be easily excitedby a radiate excitation source due to their non-localized electrons.

Advantageously, such sealing or cover layers will exhibit low naturalfluorescence at the excitation and detection wavelength used to detectthe biomaterial; will be dimensionally stable and not flow into anymicrofluidic channels present; will adhere to the base plate withoutcreating voids or gaps that may allow migration of the components fromone channel to an adjacent channel; is compatible with the chemicalreagents used in the microchannels and reservoirs such aselectrophoretic media and biomaterials including DNA fragments andpolypeptides; be compatible with the separation conditions employedincluding pH (e.g., pH of 2-12, preferably 3-8), electric fieldpotentials and voltage gradients of 200 volts/cm; exhibit good stabilityto moisture and temperature change; preferably contain no chargedsubstituents which may interfere with the separation of biomaterialsthat contain charged groups; contain no leachable components that maycontaminate the sample; and exhibit little or no spectral emission inthe wavelength range of 400 to 800 nM. Such sealing or cover layers maypossess pressure sensitive adhesive properties or be heat sealable.

With respect to the material used in the assay platform, such materialmay be either flexible or rigid, but is preferable that such materialsbe clear and colorless; chemically compatible with electrophoreticseparation; exhibit little or no fluorescence under assay detectionconditions as evidenced by little or no spectral emissions in thewavelength of 400 to 800 nM; be dimensionally stable and withstandpressure during electrophoresis; and dissipate heat duringelectrophoresis; and have minimal cross-sectional dimension.

When the sealing layer comprises a pressure sensitive adhesive or is apolymeric layer which adheres to itself, it is preferable to use a linerto protect the sealing surface. If a liner is used, it is desirable thatthere is no transfer of compounds from the liner to the sealing surfacewhich will interfere with the separation of biomaterials or increase thefluorescence of the sealing surface.

Materials suitable for use in the present invention which exhibitminimal or low fluorescence and which may be used as substrate materialsinclude but are not limited to polyolefins, polysiloxanes,polyalkylmethacrylates, and polycarbonates. Examples of suitablesubstrate films include Rohm PLEXIGLASS S30K, Rohm OROGLASS, andGoodfellow Polymethylmethacrylate and Mitsubishi SKINKOLITE HBS 007.

Materials suitable for use in the present invention as sealing or covermaterials which exhibit minimal or low fluorescence include but are notlimited to the above materials as well as adhesives such asalkyl(meth)acrylic acid esters. Preferred adhesive compositions containno aromatic moieties such as those found in aromatic solvents, aromaticmonomers, polymerization inhibitors or polymerization initiators as thepresence of the aromatic moiety will result in undesirable spectralemissions. If any aromatic solvents such as toluene or xylene are usedin the formation of the adhesive, they are removed during the dryingprocess or by subsequent treatment of the product. Silicone-basedadhesives such as Dow 7657 or Sylgard 184 (polydimethyl siloxane) mayalso be used. A low fluorescence sealing layer may be provided withadvantage comprised of amorphous polyolefins such as polyethylene,polypropylene or blends of polyolefins.

Preferred acrylate-based pressure sensitive adhesives are formulatedusing alkyl (alkyl)acrylate esters that are polymerized usingnon-aromatic initiator and cross-linkers. The concentration of unreactedcomponents such a monomers, initiators, and crosslinkers should beminimized to insure low fluorescence. Typically, the concentration ofunreacted monomers in the formulation will be in the ppm range When anorganic solvent is used in the formulation, non-aromatic solvents suchas low molecular weight hydrocarbons, alcohols, and esters arepreferred. Solvents such as heptane, hexane, ethyl acetate andisopropanol are preferred. Reactive monomers such as acrylic acid may beinhibited using substituted hydroquinones. Substituted hydroquinonesextend the shelf life of the reactive monomers and higher concentrationsare used for the most reactive monomers. Substituted hydroquinones inthe sealing layer may fluoresce significantly after exposure to radiantenergy due to their low activation energy.

1. In a microfluidic diagnostic device comprised of a base having atleast one fluid channel within which an aqueous fluid sample to beassayed passes from an inlet port to a detection zone, the improvementcomprising said at least one fluid channel being enclosed by at leastone enclosure surface wherein at least one surface of the fluid channelcomprises either a hydrophilic heat-sealable adhesive or a hydrophilicpressure sensitive adhesive, wherein the hydrophilic adhesive increasesthe surface energy of a fluid flow path through the channel and whereinthe hydrophilic adhesive decreases the surface tension of aqueous fluidflowing through the channel.
 2. The microfluidic device of claim 1,wherein said enclosure surface is heat-sealed to said base to seal saidat least one fluid channel by the hydrophilic heat-sealable adhesive. 3.The microfluidic device of claim 1, wherein the surface of saidenclosure surface comprises a pressure sensitive adhesive.
 4. Themicrofluidic device of claim 1, wherein said enclosure surface iscomprised of a low fluorescent material.
 5. The microfluidic device ofclaim 1, wherein said enclosure surface is comprised of a polymericmaterial with which a surfactant is admixed to provide hydrophilicsurface properties.
 6. The microfluidic device of claim 1, wherein saidenclosure surface is comprised of a polymeric material having asurfactant applied to a surface thereof.
 7. The microfluidic device ofclaim 5, wherein said surfactant is a non-ionic or anionic surfactant.8. The microfluidic device of claim 7, wherein said surfactant isselected from the group consisting of polyethylene oxide, polypropyleneoxide, nonylphenol ethyoxylate and polyalkylene-oxide modifiedheptamethyltrisiloxane.
 9. The microfluidic device of claim 7, whereinsaid surfactant is selected from the group consisting of sodium orammonium salts of nonyl phenol ethoxyl sulfonic acid, sodium laurylsulfate, sodium 2-ethylhexyl sulfate and sodium dioctylsulfo succinate.10. The microfluidic device of claim 7, wherein said surfactant isselected from the group consisting of the ionic salt of2-acrylamido-2-methyl propanesulfonic acid, N-vinyl caprolactam,caprolactone acrylate, N-vinyl pyrrolidone, and sulfate and acrylicmonomers.
 11. A detection device comprising: a substrate providing asurface to carry an aqueous fluid from a first end of the substratetoward a detection zone along a fluid flow path, a portion of thesubstrate surface comprising a hydrophilic heat-sealable adhesive or ahydrophilic pressure sensitive adhesive wherein the hydrophilic adhesiveincreases the surface energy of the fluid flow path across the portionof the substrate surface comprising the hydrophilic adhesive and whereinthe hydrophilic adhesive decreases the surface tension of the aqueousfluid flowing along the fluid flow path across the portion of thesubstrate surface comprising the hydrophilic adhesive.
 12. The device ofclaim 11, wherein the hydrophilic adhesive comprises a polymericmaterial and at least one surfactant, wherein the surfactant increasesthe hydrophilicity of the polymeric material.
 13. The device of claim12, wherein the surfactant is selected from the group consisting ofsodium salt of sulfated nonylphenol ethoxylate, ammonium salt ofsulfated nonylphenol ethoxylate, sodium 2-ethyl hexyl sulfate, sodiumoctyl sulfate, sodium nonylphenol sulfate, nonyl phenol ethoxylate,sodium lauryl sulfate, sodium nonylphenol ether sulfate, polyalkyleneoxide modified heptamethyl trisiloxane, sodium dioctyl sulfo succinate,and sodium salt of 2-acrylamide-2-methyl-propane sulfonic acid.
 14. Themicrofluidic device of claim 5, wherein said surfactant is selected fromthe group consisting of sodium salt of sulfated nonylphenol ethoxylate,ammonium salt of sulfated nonylphenol ethoxylate, sodium 2-ethyl hexylsulfate, sodium octyl sulfate, sodium nonylphenol sulfate, nonyl phenolethoxylate, sodium lauryl sulfate, sodium nonylphenol ether sulfate,polyalkylene oxide modified heptamethyl trisiloxane, sodium dioctylsulfo succinate, and sodium salt of 2-acrylamide-2-methyl-propanesulfonic acid.
 15. The microfluidic device of claim 6, wherein saidsurfactant is selected from the group consisting of sodium salt ofsulfated nonylphenol ethoxylate, ammonium salt of sulfated nonylphenolethoxylate, sodium 2-ethyl hexyl sulfate, sodium octyl sulfate, sodiumnonylphenol sulfate, nonyl phenol ethoxylate, sodium lauryl sulfate,sodium nonylphenol ether sulfate, polyalkylene oxide modifiedheptamethyl trisiloxane, sodium dioctyl sulfo succinate, and sodium saltof 2-acrylamide-2-methyl-propane sulfonic acid.